Use of Polysaccharide for Treating ST-Elevation Myocardial Infarction and Diseases of the Digestion System

ABSTRACT

Provided herein are methods of treating ST-elevation myocardial infarction (STEMI) and diseases of the digestion system using β-1,3/1,6-D-glucan derived from mycellium extract of  Ganoderma lucidum . Methods of treating ST-elevation myocardial infarction (STEMI) and diseases of the digestion system using a polysaccharide comprising β-1,3/1,6-D-glucan are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 62/419,174, filed Nov. 8, 2016, and 62/553,647, filed Sep. 1, 2017,the disclosure of each of which is incorporated herein by reference inits entirety.

TECHNICAL FIELD

This invention relates to the therapeutic treatment of ST-elevationmyocardial infarction (STEMI) and diseases of the digestion system usingβ-1,3/1,6-D-glucan derived from mycellium extract of Ganoderma lucidum.

BACKGROUND

Heart disease is one of the leading causes of mortality globally. Everyyear in the US 610,000 people die of heart disease, which isapproximately 1 in 4 deaths. Coronary heart disease (CHD) is the mostcommon type of heart disease, killing over 370,000 people annually (seee.g., Underlying Cause of Death 1999-2013 on CDC WONDER Online Database,released 2015). Factors such as smoking, fat and cholesterol level inthe blood, hypertension, obesity, and high blood glucose level due todiabetes play an important role in the occurrence of coronary heartdisease. When heart damage occurs, the body begins the healing processof the wounds by forming plaque in the injured arteries. Additionalfactors may also increase the risk of atherosclerosis, for example highlevels of blood cholesterol, high blood pressure, smoking, insulinresistance, diabetes, obesity, and lack of physical activity.

SUMMARY

The present application provides, inter alia, methods of treating adisease of the digestive system in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof.

In some embodiments, the disease of the digestive system comprisesinflammation of one or more organs of the digestive system. In someembodiments, the disease of the digestive system is selected from thegroup consisting of inflammatory bowel disease, Crohn's disease, andulcerative colitis.

The present application further provides methods of treatingST-elevation myocardial infarction in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof.

In some embodiments, the methods comprise administering about 500 mg toabout 600 mg β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, to the subject. In some embodiments, the methods compriseadministering about 500 mg to about 550 mg β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, to the subject.

The present application further provides methods of treating a diseaseof the digestive system in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition comprisingβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, andone or more pharmaceutically acceptable excipients.

The present application further provides methods of treatingST-elevation myocardial infarction in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical composition comprises about 500mg to about 600 mg of the β-1,3/1,6-D-glucan, or a pharmaceuticallyacceptable salt thereof. In some embodiments, the pharmaceuticalcomposition comprises about 500 mg to about 550 mg of theβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof.

The present application further provides methods of treatinginflammatory bowel disease in a subject in need thereof, comprisingadministering to the subject about 500 mg to about 600 mg ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof.

The present application further provides methods of treating Crohn'sdisease in a subject in need thereof, comprising administering to thesubject about 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof.

The present application further provides methods of treating ulcerativecolitis in a subject in need thereof, comprising administering to thesubject about 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof.

The present application further provides methods of treatingST-elevation myocardial infarction in a subject in need thereof,comprising administering to the subject about 500 mg to about 600 mg ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof.

The present application further provides methods of treatinginflammatory bowel disease in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition comprisingabout 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.

The present application further provides methods of treating Crohn'sdisease in a subject in need thereof, comprising administering to thesubject a pharmaceutical composition comprising about 500 mg to about600 mg of β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients.

The present application further provides methods of treating ulcerativecolitis in a subject in need thereof, comprising administering to thesubject a pharmaceutical composition comprising about 500 mg to about600 mg of β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients.

The present application further provides methods of treatingST-elevation myocardial infarction in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising about 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.

The present application further provides methods of treating one or moresymptoms of a disease of the digestive system in a subject in needthereof, comprising: a) identifying or having identified a subject asexhibiting one or more symptoms of the disease of the digestive system;and b) administering to the subject a therapeutically effective amountof β-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof.

In some embodiments, the subject is identified or has been identified asexhibiting inflammation associated with a disease of the digestivesystem. In some embodiments, the subject is identified or has beenidentified as exhibiting one or more symptoms associated with a diseaseof the digestive system selected from the group consisting ofinflammatory bowel disease, Crohn's disease, and ulcerative colitis.

The present application further provides methods of treating one or moresymptoms of ST-elevation myocardial infarction in a subject in needthereof, comprising: a) identifying or having identified a subject asexhibiting one or more symptoms of the ST-elevation myocardialinfarction; and b) administering to the subject a therapeuticallyeffective amount of β-1,3/1,6-D-glucan, or a pharmaceutically acceptablesalt thereof.

In some embodiments, the methods comprise administering about 500 mg toabout 600 mg β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, to the subject. In some embodiments, the method comprisesadministering about 500 mg to about 550 mg β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, to the subject.

The present application further provides methods of treating one or moresymptoms of a disease of the digestive system in a subject in needthereof, comprising: a) identifying or having identified a subject asexhibiting one or more symptoms of the disease of the digestive system;and b) administering to the subject a pharmaceutical compositioncomprising β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients.

In some embodiments, the subject is identified or has been identified asexhibiting inflammation associated with a disease of the digestivesystem. In some embodiments, the subject is identified or has beenidentified as exhibiting one or more symptoms associated with a diseaseof the digestive system selected from the group consisting ofinflammatory bowel disease, Crohn's disease, and ulcerative colitis.

The present application further provides methods of treating one or moresymptoms of ST-elevation myocardial infarction in a subject in needthereof, comprising: a) identifying or having identified a subject asexhibiting one or more symptoms of the ST-elevation myocardialinfarction; and b) administering to the subject a pharmaceuticalcomposition comprising β-1,3/1,6-D-glucan, or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptableexcipients.

In some embodiments, the pharmaceutical composition comprises about 500mg to about 600 mg of the β-1,3/1,6-D-glucan, or a pharmaceuticallyacceptable salt thereof. In some embodiments, the pharmaceuticalcomposition comprises about 500 mg to about 550 mg of theβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof.

In some embodiments, the methods further comprise identifying or havingidentified the subject as exhibiting abnormal expression of one or morecytokines. In some embodiments, the subject is identified or has beenidentified as exhibiting abnormal expression of interleukin-6,interleukin-10, tumor necrosis factor-α, C-reactive protein, or anycombination thereof.

In some embodiments, the methods further comprise administering one ormore additional therapeutic agents.

In some embodiments, about 5 mg/kg/day to about 10 mg/kg/day of theβ-1,3/1,6-D-glucan is administered to the subject. In some embodiments,about 7 mg/kg/day to about 10 mg/kg/day of the β-1,3/1,6-D-glucan isadministered to the subject.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

DESCRIPTION OF DRAWINGS

FIG. 1 shows representative illustration of the lamina propria in ahealthy subject (Panel A) and several cellular events which contributeto increased bacterial adherence to epithelial cells (Panel B).

FIGS. 2A-2B show a representative size exclusion chromatograph profileof the polysaccharide isolated from Ganoderma lucidum mycelium,comprising β-1,3/1,6-D-glucan.

FIG. 3 shows the glycosyl composition analysis of the polysaccharidecomprising β-1,3/1,6-D-glucan isolated from Ganoderma lucidum mycelium.

FIG. 4 shows the glycosyl linkage analysis of the polysaccharidecomprising β-1,3/1,6-D-glucan isolated from Ganoderma lucidum mycelium.

FIG. 5 shows 1D-Proton NMR spectrum of the Pk 1 sample in DMSO-d₆:D₂O(6:1 mixture).

FIG. 6A-6B shows the effect of β-1,3/1,6-D-glucan administration on thelevel of inflammatory parameters Interleukin-6 (IL-6) (FIG. 6A), tumornecrosis factor-α (TNF-α) and high-sensitive C-reactive protein (hs-CRP)(FIG. 6B) in patients with stable angina pectoris (SAP).

FIG. 7 shows the effect of β-1,3/1,6-D-glucan administration on MDA, SODand NO levels in patients with stable angina pectoris (SAP).

FIG. 8 shows the effect of β-1,3/1,6-D-glucan administration on totalcholesterol, low-density lipid (LDL), triglyceride (TG), andhigh-density lipid (HDL) levels in patients with stable angina pectoris(SAP).

FIG. 9 shows the effect of β-1,3/1,6-D-glucan administration on thelevel of endothelial dysfunction parameters, circulating endothelialcell (CEC) and endothelial progenitor cell (EPC) in patients with stableangina pectoris (SAP).

FIG. 10 shows the effect of β-1,3/1,6-D-glucan administration on HbA1clevels in patients with stable angina pectoris (SAP).

FIG. 11A-11B shows the effect of β-1,3/1,6-D-glucan administration onthe level of inflammatory parameters Interleukin-6 (IL-6) (FIG. 11A),tumor necrosis factor-α (TNF-α) and high-sensitive C-reactive protein(hs-CRP) (FIG. 11B) in high risk patients.

FIG. 12 shows the effect of β-1,3/1,6-D-glucan administration on MDA,SOD and NO levels in high risk patients.

FIG. 13 shows the effect of β-1,3/1,6-D-glucan administration on totalcholesterol, low-density lipid (LDL), triglyceride (TG), andhigh-density lipid (HDL) levels in high risk patients.

FIG. 14 shows the effect of β-1,3/1,6-D-glucan administration on thelevel of endothelial dysfunction parameters, circulating endothelialcell (CEC) and endothelial progenitor cell (EPC) in high risk patients.

FIG. 15 shows the effect of β-1,3/1,6-D-glucan administration on HbA1clevels in high risk patients.

FIG. 16 shows a flow chart describing a treatment regimen of patientshaving STEMI by administration of β-1,3/1,6-D-glucan.

DETAILED DESCRIPTION

Ganoderma lucidum is a genus of polypore fungus that grows on wood andconsists of about 80 species, which are generally found in tropicalenvironments (Krik et al, Dictionary of the Fungi, 10^(th) Ed, CABIEurope, 2008). Several classes of bioactive substances isolated andidentified from G. lucidum such as triterpenoids, polysaccharides,nucleosides, sterols, and alkaloids have been shown to function asanti-inflammatory agents, anti-tumor agents, antioxidant agents,immunomodulators, and radioprotection agents (see e.g., Xu et al,American Journal of Chinese Medicine, 2011, 39(1):15-27). The presentapplication provides polysaccharides, derived from Ganoderma lucidummycelium which comprise β-1,3/1,6-D-glucan as an active component (e.g.,an active component towards treating the diseases provided herein).β-D-glucans are major components of Ganoderma mycelium, which, in thespores of Ganoderma, is a minor component (Chan et al, Journal ofHematology & Oncology, 2009, 2:25).

Previous studies regarding the function and role of bioactivepolysaccharides have demonstrated that β-1,3/1,6-D-glucan exhibitsproperties including anti-inflammatory, anti-tumor, antioxidant, andimmunomodulating radioprotection (see e.g., Xu et al, American Journalof Chinese Medicine, 2011, 39(1):15-27). For example, orallyadministered β-glucans have been shown to be taken up (i.e.,phagocytized) by macrophages in Peyer Patch's via the Dectin-1 receptorand were subsequently transported to the spleen, lymph nodes, and bonemarrow. Within the bone marrow, the macrophages defragmented the largeβ-1,3-glucan molecule into smaller soluble β-1,3-glucan fragments. Thesmall β-glucan fragments were then released by the macrophages and takenup by the circulating granulocytes, monocytes and dendritic cells.Despite low systemic blood levels (less than 0.5%), significant systemicimmunomodulating effects in terms of humoral and cellular immuneresponses were demonstrated (see e.g., Chan et al, Journal of Hematology& Oncology, 2009, 2(25):1-11). In addition, β-1,3/1,6-D-glucan effects avariety of membrane receptors found in the immune cells such as:Dectin-1, Complement Receptor-3 (CR-3), Toll-Like Receptor (TLR),Scavenger Receptor, Lactosylceramide Receptor and Langerin Receptor (seee.g., Chan et al, Journal of Hematology & Oncology, 2009, 2(25):1-11;and Clark et al, Biology and Chemistry of β-Glucan, 2011, 1:19-38) Anumber of reports have demonstrated that Ganoderma lucidumPolysaccharide Peptide (GLPP) modulates immune function both in vivo andin vitro. The immuno-modulating effects of GLPP are extensive, includingpromoting the function of Antigen Presenting-Cell (APC), mononuclearphagocyte system, humoral and cellular immunity (see e.g., Lin et al, J.Pharmacol. Sci. 2005, 99:144-153), which is useful for treatment of thediseases provided herein.

In addition, the β-1,3/1,6-D-glucan described herein can increase theactivity of superoxide dismutase (SOD) and catalase (CAT) (Jia et al,Food Chemistry, 2008, 115:32-36), which is useful for treatment of thedisease provided herein. Cellular oxidative damage has been known as amajor cause of cells and tissues injury and is primarily caused byReactive Oxygen Species (ROS) that binds to many normal cell components,reacts with unsaturated lipid membranes, denatures the protein andattacks nucleic acid. As a result of the active metabolic oxygenformation imbalance and an elimination of both enzymatic andnon-enzymatic antioxidants, this process is called an oxidative stress.Oxidative stress plays an important role in physiological conditions andalso various diseases such as diabetes mellitus, myocardial infarctionand carcinogenesis. The cells in the body possess antioxidant mechanismsuch as glucose-6-phosphate dehydrogenase, superoxide dismutase (SOD),catalase (CAT), glutathione-S-transferase (GST), which are useful toprevent the formation of free radicals and repair of oxidative damage.SOD and CAT are major enzymes that eliminate the poison of free radicalscavenger of in vivo. SOD enzyme adopts superoxide radicals (02) andcatalyzes the conversion of these two radicals to form molecularhydrogen and oxygen peroxidase. Hydrogen peroxidase is formed by SOD andother processes by CAT. Additional uses of the β-1,3/1,6-D-glucandescribed herein are described, for example, in Appendix A of U.S.Provisional Patent Application No. 62/553,647, the disclosure of whichis incorporated herein by reference in its entirety.

In some embodiments, the polysaccharide comprises about 30% to about 80%total glucan, for example, about 30% to about 80%, about 30% to about70%, about 30% to about 60%, about 30% to about 50%, about 30% to about40%, about 40% to about 80%, about 40% to about 70%, about 40% to about60%, about 40% to about 50%, about 50% to about 80%, about 50% to about70%, about 50% to about 60%, about 60% to about 80%, about 60% to about70%, or about 70% to about 80% total glucan.

In some embodiments, the molecular weight of the polysaccharide is fromabout 3600 kDa to about 4200 kDa, for example, about 3600 kDa to about4200 kDa, about 3800 kDa to about 4200 kDa, about 4000 kDa to about 4200kDa, about 3800 kDa to about 4200 kDa, about 4000 kDa to about 4200 kDa,or about 4000 kDa to about 4200 kDa.

In some embodiments, the total glucan comprises β-glucan and α-glucan.In some embodiments, the total glucan consists essentially of β-glucan.In some embodiments, the total glucan comprises greater than about 90%β-glucan, greater than about 95% β-glucan, greater than about 96%β-glucan, greater than about 97% β-glucan, greater than about 98%β-glucan, greater than about 99% β-glucan, greater than about 99.1%β-glucan, greater than about 99.2% β-glucan, greater than about 99.3%β-glucan, greater than about 99.4% β-glucan, greater than about 99.5%β-glucan, greater than about 99.6% 3-glucan, greater than about 99.7%β-glucan, greater than about 99.8% β-glucan, or greater than about 99.9%β-glucan.

In some embodiments, the ratio of 1,3- to 1,6-glucan linkages in theβ-1,3/1,6-D-glucan is from about 1:1 to about 3:1. In some embodiments,the ratio of 1,3- to 1,6-glucan linkages in the β-1,3/1,6-D-glucan isabout 2:1.

In some embodiments, the glycosyl composition of the polysaccharidecomprising the β-1,3/1,6-D-glucan comprises saccharides is selected fromthe group consisting of fucose, xylose, mannose, galactose, and glucose.In some embodiments, the glycosyl composition of the polysaccharide isas shown in Table 1B. In some embodiments, the glycosyl linkages of thepolysaccharide is as shown in Table 2. In some embodiments, thepolysaccharide consists essentially of β-1,3/1,6-D-glucan. In someembodiments, the glycosyl composition of the polysaccharide is as shownin Table 1B and the glycosyl linkages of the polysaccharide are as shownin Table 2.

In some embodiments, the polysaccharide comprises one or more terminalfucopyranosyl groups, one or more terminal xylopyranosyl groups, one ormore terminal mannopyranosyl groups, one or more terminal glucopyranosylgroups, one or more 2-xylopyranosyl groups, one or more 3-glucopyranosylgroups, one or more 2-mannopyranosyl groups, one or more 3-linkedmannopyranosyl groups, one or more 2-glucopyranosyl groups, one or more4-mannopyranosyl groups, one or more 6-glucopyranosyl groups, one ormore 4-glucopyranosyl groups, one or more 2,3-mannopyranosyl groups, oneor more 3,4-mannopyranosyl groups, one or more 3,4-glucopyranosylgroups, one or more 2,3-glucopyranosyl groups, one or more2,4-glucopyranosyl groups, one or more 3,6-glucopyranosyl groups, one ormore 4,6-galactopyranosyl groups, one or more 2,3,4-glucopyranosylgroups, one or more 3,4,6-glucopyranosyl groups, and one or more2,3,6-glucopyranosyl groups.

In some embodiments, the polysaccharide consists essentially of one ormore terminal fucopyranosyl groups, one or more terminal xylopyranosylgroups, one or more terminal mannopyranosyl groups, one or more terminalglucopyranosyl groups, one or more 2-xylopyranosyl groups, one or more3-glucopyranosyl groups, one or more 2-mannopyranosyl groups, one ormore 3-linked mannopyranosyl groups, one or more 2-glucopyranosylgroups, one or more 4-mannopyranosyl groups, one or more6-glucopyranosyl groups, one or more 4-glucopyranosyl groups, one ormore 2,3-mannopyranosyl groups, one or more 3,4-mannopyranosyl groups,one or more 3,4-glucopyranosyl groups, one or more 2,3-glucopyranosylgroups, one or more 2,4-glucopyranosyl groups, one or more3,6-glucopyranosyl groups, one or more 4,6-galactopyranosyl groups, oneor more 2,3,4-glucopyranosyl groups, one or more 3,4,6-glucopyranosylgroups, and one or more 2,3,6-glucopyranosyl groups.

In some embodiments, the polysaccharide consists of one or more terminalfucopyranosyl groups, one or more terminal xylopyranosyl groups, one ormore terminal mannopyranosyl groups, one or more terminal glucopyranosylgroups, one or more 2-xylopyranosyl groups, one or more 3-glucopyranosylgroups, one or more 2-mannopyranosyl groups, one or more 3-linkedmannopyranosyl groups, one or more 2-glucopyranosyl groups, one or more4-mannopyranosyl groups, one or more 6-glucopyranosyl groups, one ormore 4-glucopyranosyl groups, one or more 2,3-mannopyranosyl groups, oneor more 3,4-mannopyranosyl groups, one or more 3,4-glucopyranosylgroups, one or more 2,3-glucopyranosyl groups, one or more2,4-glucopyranosyl groups, one or more 3,6-glucopyranosyl groups, one ormore 4,6-galactopyranosyl groups, one or more 2,3,4-glucopyranosylgroups, one or more 3,4,6-glucopyranosyl groups, and one or more2,3,6-glucopyranosyl groups.

In some embodiments, the polysaccharide comprising theβ-1,3/1,6-D-glucan provided herein, or pharmaceutically acceptable saltthereof, is substantially isolated. The term “substantially isolated”means that the polysaccharide is at least partially or substantiallyseparated from the environment in which it was formed or detected.Partial separation can include, for example, a composition enriched inthe compounds provided herein. Substantial separation can includecompositions containing at least about 50%, at least about 60%, at leastabout 70%, at least about 80%, at least about 90%, at least about 95%,at least about 97%, or at least about 99% by weight of the compoundsprovided herein, or salt thereof. General methods for isolatingcompounds and their salts are routine in the art. In some embodiments,the polysaccharide is isolated according to the methods described inIndonesian Patent Application No. P-00 2011 00579, the disclosure ofwhich is incorporated herein by reference in its entirety. In someembodiments, the polysaccharide comprising the β-1,3/1,6-D-glucanfurther comprises α-1,3/1,6-D-glucan.

The present invention further provides use of the β-1,3/1,6-D-glucandescribed herein, or a pharmaceutically acceptable salt thereof, for thepreparation of a medicament for use in any of the methods describedherein.

ST-Elevation Myocardial Infarction (STEMI)

The present application provides, inter alia, methods of treatingST-elevation myocardial infarction (STEMI) in a subject in need thereof,comprising administering to the subject a therapeutically effectiveamount of β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof. In some embodiments, β-1,3/1,6-D-glucan is administered to thesubject. In some embodiments, the polysaccharide comprising theβ-1,3/1,6-D-glucan is administered to the subject.

STEMI usually occurs when the coronary blood flow decreases abruptlyafter thrombus occlusion on the preexisted arteriosclerotic plaque. Forexample, STEMI occurs when coronary artery thrombosis occurs rapidly atthe site of vascular injury, where the injury may be exacerbated byfactors such as smoking, hypertension, and lipid accumulation. In manycases, infarction occurs when atherosclerotic plaque experiencesfissures, rupture, or ulceration. If local or systemic conditionstrigger thrombogenesis, mural thrombus will develop on the location ofthe rupture, resulting in coronary artery occlusion. Histologicalstudies have shown that coronary plaque is prone to rupture if it has athin fibrous cap and a lipid-rich core. In STEMI, classic pathologicalfeatures include a fibrin rich red thrombus, which without being boundby theory, is believed to be one of the reasons why STEMI responds tothrombolytic therapy. At the location of plaque rupture, variousagonists (e.g., collagen, ADP, epinephrine, serotonin) trigger theactivation of platelets, which in turn produce and release thromboxaneA2 (a local potent vasoconstrictor). The activation of thrombosis alsogenerates a conformational change in the receptor glycoprotein IIb/IIIa.After experiencing a conversion on its function, the receptor has a highaffinity for the amino acid sequence in a soluble adhesion protein(integrins) such as von Willebrand factor (vWF) and fibrinogen, both ofwhich are multivalent molecules that can bind two different plateletssimultaneously, resulting in a cross linking of platelets andaggregation. The cascade coagulation is activated by the exposure oftissue factor in the damaged endothelial cells. Activated Factors VIIand X will result in the conversion of prothrombin into thrombin, whichthen will convert fibrinogen to fibrin. The involved coronary arterieswill then experience an occlusion by thrombus composed of platelet andfibrin aggregates. In some circumstances, STEMI may also result fromcoronary embolism, congenital abnormalities, coronary spasm, and varioussystemic inflammatory diseases (see e.g., Alwi et al, Internal Medicine,vol. II, ed. VI, Ch. 191; pp: 1459-1460; InternaPublishing, Jakarta(2014)).

The present application further provides methods of treating one or moresymptoms of ST-elevation myocardial infarction in a subject in needthereof, comprising: a) identifying or having identified a subject asexhibiting one or more symptoms of the ST-elevation myocardialinfarction; and b) administering to the subject a therapeuticallyeffective amount of β-1,3/1,6-D-glucan, or a pharmaceutically acceptablesalt thereof.

In some embodiments, the method comprises administering about 10 mg toabout 1 g β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, to the subject, for example, about 10 mg to about 1 g, about 10mg to about 800 mg, about 10 mg to about 600 mg, about 10 mg to about400 mg, about 10 mg to about 200 mg, about 10 mg to about 100 mg, about10 mg to about 50 mg, about 50 mg to about 1 g, about 50 mg to about 800mg, about 50 mg to about 600 mg, about 50 mg to about 400 mg, about 50mg to about 200 mg, about 50 mg to about 100 mg, about 100 mg to about 1g, about 100 mg to about 800 mg, about 100 mg to about 600 mg, about 100mg to about 400 mg, about 100 mg to about 200 mg, about 200 mg to about1 g, about 200 mg to about 800 mg, about 200 mg to about 600 mg, about200 mg to about 400 mg, about 400 mg to about 1 g, about 400 mg to about800 mg, about 400 mg to about 600 mg, about 600 mg to about 1 g, about600 mg to about 800 mg, or about 800 mg to about 1 g. In someembodiments, the method comprises administering about 500 mg to about600 mg β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, to the subject. In some embodiments, the method comprisesadministering about 500 mg to about 550 mg β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, to the subject.

The present application further provides methods of treatingST-elevation myocardial infarction in a subject in need thereof,comprising administering to the subject about 500 mg to about 600 mg ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof. Insome embodiments, about 500 mg to about 600 mg of β-1,3/1,6-D-glucan isadministered to the subject.

In some embodiments, about 1 mg/kg/day to about 20 mg/kg/day of theβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, isadministered to the subject, for example, about 1 mg/kg/day to about 20mg/kg/day, about 1 mg/kg/day to about 15 mg/kg/day, about 1 mg/kg/day toabout 10 mg/kg/day, about 1 mg/kg/day to about 5 mg/kg/day, about 5mg/kg/day to about 20 mg/kg/day, about 5 mg/kg/day to about 15mg/kg/day, about 5 mg/kg/day to about 10 mg/kg/day, about 10 mg/kg/dayto about 20 mg/kg/day, about 10 mg/kg/day to about 15 mg/kg/day, orabout 15 mg/kg/day to about 20 mg/kg/day. In some embodiments, about 5mg/kg/day to about 10 mg/kg/day of the β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, is administered to thesubject. In some embodiments, about 5 mg/kg/day to about 10 mg/kg/day ofthe β-1,3/1,6-D-glucan is administered to the subject. In someembodiments, about 7 mg/kg/day to about 10 mg/kg/day of theβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, isadministered to the subject. In some embodiments, about 7 mg/kg/day toabout 10 mg/kg/day of the β-1,3/1,6-D-glucan is administered to thesubject.

In some embodiments, the methods provided herein further comprisemodulating one or more cytokines or an activity associated with one ormore cytokines in the subject, comprising contacting a cell or tissueassociated with abnormal expression of the one or more cytokines withβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof. Insome embodiments, the modulating is inhibition. In some embodiments, themodulating is activation. In some embodiments, the contacting isperformed in vitro. In some embodiments, the contacting is performed invivo. In some embodiments, the methods comprise modulating one or morecytokines selected from the group consisting of interleukin-6,interleukin-10, tumor necrosis factor-α, C-reactive protein, or anycombination thereof, in the subject. In some embodiments, the methodscomprise modulating one or more cytokine activities in the subject. Insome embodiments, the methods provided herein further compriseidentifying the subject, or having the subject identified, as exhibitingabnormal expression of one or more cytokines. In some embodiments, thesubject is identified or has been identified as exhibiting abnormalexpression of interleukin-6, interleukin-10, tumor necrosis factor-α,C-reactive protein, or any combination thereof. In some embodiments, thecell or tissue is contacted with β-1,3/1,6-D-glucan.

In some embodiments, the methods provided herein further comprisemodulating myosin in the subject, comprising contacting a cell or tissueassociated with abnormal expression of myosin with β-1,3/1,6-D-glucan,or a pharmaceutically acceptable salt thereof.

In some embodiments, the methods provided herein further comprisemodulating one or more of MDA, SOD, IL-6, hsCRP, TNF-α, totalcholesterol, triglycerides (TG), low-density lipids (LDL), high-densitylipids (HDL), fasting glucose, HbA1c, systolic BP, diastolic BP, theratio of EPC-CEC, or any combination thereof, in the subject.

Diseases of the Digestive System

Idiopathic inflammatory bowel disease comprises two types of chronicintestinal disorders, Crohn's disease (CD), and ulcerative colitis (UC).Accumulating evidence suggests that inflammatory bowel disease resultsfrom inappropriate inflammatory response to intestinal microbes in agenetically susceptible host. As shown in FIG. 1, the lamina proprianormally contains a diverse array of immune cells and secretedcytokines. These include anti-inflammatory mediators (transforminggrowth factor β [TGF-β] and interleukin-10 [IL-10]) that down-regulateimmune responses, as well as proinflammatory mediators from both innateand adaptive immune cells that limit excessive entry of intestinalmicrobiota and defend against pathogens. Non-inflammatory defenses, suchas phagocytosis by macrophages, are believed to assist in defendingagainst bacteria entering the lamina propria while minimizing tissueinjury. A homeostatic balance is maintained between regulatory T cells(e.g., Treg) and effector cells (Th1, Th2, and Th17).

Several events contribute to increased bacterial adherence to epithelialcells. In inflammatory bowel disease (IBD), innate cells produceincreased levels of Tumor Necrosis Factor α (TNF-α), interleukin-1β,interleukin-6, interleukin-12, interleukin-23, and chemokines. There ismarked expansion of the lamina propria, with increased numbers of CD4+ Tcells, especially proinflammatory T-cell subgroups, which also secreteincreased levels of cytokines and chemokines. Increased production ofchemokines results in recruitment of additional leukocytes, resulting ina cycle of inflammation.

Accordingly, the present application provides methods of treating adisease of the digestive system in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof.Example diseases of the digestive system include, but are not limitedto, inflammatory bowel disease, Crohn's disease, and ulcerative colitis.In some embodiments, β-1,3/1,6-D-glucan is administered to the subject.In some embodiments, a polysaccharide comprising β-1,3/1,6-D-glucan isadministered to the subject.

In some embodiments, the disease is associated with an abnormalinflammatory response of the digestive system. In some embodiments, thedisease is associated with an abnormal inflammatory response of one ormore organs of the digestive system (e.g., esophagus, stomach, smallintestine, and large intestine). In some embodiments, the disease isassociated with an abnormal inflammatory response of one or more organsassociated with the digestive system (e.g., tongue, salivary glands,pancreas, liver, gallbladder, and the like). In some embodiments, thedisease of the digestive system comprises inflammation of one or moreorgans of the digestive system. In some embodiments, the disease of thedigestive system comprises inflammation of one or more organs associatedwith the digestive system. In some embodiments, the disease of thedigestive system comprises inflammation of one or more organs selectedfrom the group consisting of the esophagus, stomach, small intestine,large intestine, tongue, salivary glands, pancreas, liver, andgallbladder. In some embodiments, the disease is selected from the groupconsisting of inflammatory bowel disease, Crohn's disease, andulcerative colitis, or inflammation associated with any of theaforementioned diseases.

The present application further provides methods of treating a diseaseselected from the group consisting of sepsis, idiopathicthrombocytopenic purpura (ITP), myasthenia gravis, pseudobulbar palsy,miliary tuberculosis, myocarditis, and cancer.

In some embodiments, the cancer is selected from the group consisting ofnon-small cell lung carcinoma (NSCLC) and brain cancer. In someembodiments, the cancer is a solid tumor.

In some embodiments, the methods comprise administering about 10 mg toabout 1 g β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, to the subject, for example, about 10 mg to about 1 g, about 10mg to about 800 mg, about 10 mg to about 600 mg, about 10 mg to about400 mg, about 10 mg to about 200 mg, about 10 mg to about 100 mg, about10 mg to about 50 mg, about 50 mg to about 1 g, about 50 mg to about 800mg, about 50 mg to about 600 mg, about 50 mg to about 400 mg, about 50mg to about 200 mg, about 50 mg to about 100 mg, about 100 mg to about 1g, about 100 mg to about 800 mg, about 100 mg to about 600 mg, about 100mg to about 400 mg, about 100 mg to about 200 mg, about 200 mg to about1 g, about 200 mg to about 800 mg, about 200 mg to about 600 mg, about200 mg to about 400 mg, about 400 mg to about 1 g, about 400 mg to about800 mg, about 400 mg to about 600 mg, about 600 mg to about 1 g, about600 mg to about 800 mg, or about 800 mg to about 1 g. In someembodiments, the method comprises administering about 500 mg to about600 mg β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, to the subject. In some embodiments, the method comprisesadministering about 500 mg to about 550 mg β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, to the subject.

The present application further provides methods of treatinginflammatory bowel disease in a subject in need thereof, comprisingadministering to the subject about 500 mg to about 600 mg ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof. Insome embodiments, about 500 mg to about 600 mg of β-1,3/1,6-D-glucan isadministered to the subject.

The present application further provides methods of treating Crohn'sdisease in a subject in need thereof, comprising administering to thesubject about 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof. In some embodiments, about 500mg to about 600 mg of β-1,3/1,6-D-glucan is administered to the subject.

The present application further provides methods of treating ulcerativecolitis in a subject in need thereof, comprising administering to thesubject about 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof. In some embodiments, about 500mg to about 600 mg of β-1,3/1,6-D-glucan is administered to the subject.

In some embodiments, about 1 mg/kg/day to about 20 mg/kg/day of theβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, isadministered to the subject, for example, about 1 mg/kg/day to about 20mg/kg/day, about 1 mg/kg/day to about 15 mg/kg/day, about 1 mg/kg/day toabout 10 mg/kg/day, about 1 mg/kg/day to about 5 mg/kg/day, about 5mg/kg/day to about 20 mg/kg/day, about 5 mg/kg/day to about 15mg/kg/day, about 5 mg/kg/day to about 10 mg/kg/day, about 10 mg/kg/dayto about 20 mg/kg/day, about 10 mg/kg/day to about 15 mg/kg/day, orabout 15 mg/kg/day to about 20 mg/kg/day. In some embodiments, about 5mg/kg/day to about 10 mg/kg/day of the β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, is administered to thesubject. In some embodiments, about 5 mg/kg/day to about 10 mg/kg/day ofthe β-1,3/1,6-D-glucan is administered to the subject. In someembodiments, about 7 mg/kg/day to about 10 mg/kg/day of theβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, isadministered to the subject. In some embodiments, about 7 mg/kg/day toabout 10 mg/kg/day of the β-1,3/1,6-D-glucan is administered to thesubject.

In some embodiments, the methods provided herein further comprisemodulating one or more cytokines or an activity associated with one ormore cytokines in the subject, comprising contacting a cell or tissueassociated with abnormal expression of the one or more cytokines withβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof. Insome embodiments, the modulating is inhibiting. In some embodiments, themodulating is activating. In some embodiments, the contacting isperformed in vitro. In some embodiments, the contacting is performed invivo. In some embodiments, the method comprises modulating one or morecytokines selected from the group consisting of interleukin-6,interleukin-10, tumor necrosis factor-α, C-reactive protein, or anycombination thereof, in the subject. In some embodiments, the methodsprovided herein further comprise identifying or having identified thesubject as exhibiting abnormal expression of one or more cytokines. Insome embodiments, the subject is identified or has been identified asexhibiting abnormal expression of interleukin-6, interleukin-10, tumornecrosis factor-α, C-reactive protein, or any combination thereof. Insome embodiments, the cell or tissue is contacted withβ-1,3/1,6-D-glucan.

In some embodiments, the methods provided herein further comprisemodulating myosin in the subject, comprising contacting a cell or tissueassociated with abnormal expression of myosin with β-1,3/1,6-D-glucan,or a pharmaceutically acceptable salt thereof.

In some embodiments, the methods provided herein further comprisemodulating MDA, SOD, IL-6, hsCRP, TNF-α, Total Cholesterol,Triglycerides (TG), Low Density Lipids (LDL), High Density Lipids (HDL),fasting glucose, HbA1c, systolic BP, diastolic BP, the ratio of EPC-CEC,or any combination thereof, in the subject.

Combination Therapies

One or more additional therapeutic agents such as, for example,anti-inflammatory agents, steroids, immunosuppressants, anti-plateletaggregation agents, and anesthetics (e.g., for use in combination with asurgical procedure) or other agents useful for treating cardiacdisorders (e.g., atherosclerosis, STEMI, and the like), hypertension,and diabetes (e.g., diabetes associated with heart disease), can be usedin combination with the β-1,3/1,6-D-glucan described herein fortreatment of the diseases provided herein. In some embodiments, one ormore additional therapeutic agents are administered to the subject incombination with the polysaccharide comprising the β-1,3/1,6-D-glucan.

In some embodiments, the additional therapeutic agent(s) is administeredsimultaneously with the β-1,3/1,6-D-glucan provided herein. In someembodiments, the additional therapeutic agent is administered afteradministration of the β-1,3/1,6-D-glucan provided herein. In someembodiments, the additional therapeutic agent is administered prior toadministration of the β-1,3/1,6-D-glucan provided herein.

In some embodiments, the additional therapeutic agent is administeredsimultaneously with the polysaccharide comprising the β-1,3/1,6-D-glucanprovided herein. In some embodiments, the additional therapeutic agentis administered after administration of the polysaccharide comprisingthe β-1,3/1,6-D-glucan provided herein. In some embodiments, theadditional therapeutic agent is administered prior to administration ofthe polysaccharide comprising the β-1,3/1,6-D-glucan provided herein.

Example anti-inflammatory agents include, but are not limited to,aspirin, choline salicylates, celecoxib, diclofenac potassium,diclofenac sodium, diclofenac sodium with misoprostol, diflunisal,etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, meclofenamatesodium, mefenamic acid, nabumetone, naproxen, naproxen sodium,oxaprozin, piroxican, rofecoxib, salsalate, sodium salicylate, sulindac,tolmetin sodium, and valdecoxib.

Example steroids include, but are not limited to, corticosteroids suchas cortisone, dexamethasone, hydrocortisone, methylprednisolone,prednisolone, and prednisone.

Example immunosuppressants include, but are not limited to,azathioprine, chlorambucil, cyclophosphamide, cyclosporine, daclizumab,infliximab, methotrexate, and tacrolimus.

Example agents for treating cardiac disorders include, but are notlimited to, angiotensin-converting enzyme (ACE) inhibitors (e.g.,enalapril, captopril), angiotensin II receptor blockers (e.g., losartan,valsartan), beta blockers (e.g., carvedilol, metoprolol, bisoprolol),diuretics (e.g., furosemide), aldosterone antagonists (e.g.,spironolactone, eplerenone), and digoxin (i.e., digitalis). In someembodiments, the methods provided herein further comprise administeringdigoxin to the subject.

Example agents for treating hypertension include, but are not limitedto, thiazide diuretics (e.g., hydrochlorothiazide, chlorthalidone),angiotensin-converting enzyme (ACE) inhibitors (e.g., enalapril,captopril), angiotensin II receptor blockers (e.g., losartan,valsartan), beta blockers (e.g., carvedilol, metoprolol, bisoprolol),calcium channel blockers (e.g., amlodipine, diltiazem), and renininhibitors (e.g., aliskiren). Example agents for treating diabetesinclude, but are not limited to, insulin, biguanidine agents (e.g.,metformin, phenformin, and buformin), thiazolidinediones (e.g.,pioglitazone), sulfonylureas (e.g., tolbutamide, acetohexamide,glipizide, glibenclamide, meglitinides (e.g., repaglinide, nateglinide),and alpha-glucosidase inhibitors (e.g., miglitol, acarbose, voglibose).

Example anesthetics include, but are not limited, to local anesthetics(e.g., lidocaine, procain, ropivacaine) and general anesthetics (e.g.,desflurane, enflurane, halothane, isoflurane, methoxyflurane, nitrousoxide, sevoflurane, mmobarbital, methohexital, thiamylal, thiopental,diazepam, lorazepam, midazolam, etomidate, ketamine, propofol,alfentanil, fentanyl, remifentanil, buprenorphine, butorphanol,hydromorphone levorphanol, meperidine, methadone, morphine, nalbuphine,oxymorphone, pentazocine).

In some embodiments, the β-1,3/1,6-D-glucan is administered during atherapeutic procedure (e.g., a surgical procedure, administration of oneor more additional therapeutic agents, and the like). In someembodiments, the β-1,3/1,6-D-glucan is administered in combination withan additional therapeutic agent during a therapeutic procedure.

In some embodiments, the β-1,3/1,6-D-glucan is administered from about30 minutes to about 48 hours prior to a therapeutic procedure, forexample, from about 30 minutes to about 48 hours, about 30 minutes toabout 24 hours, about 30 minutes to about 12 hours, about 30 minutes toabout 6 hours, about 30 minutes to about 2 hours, about 30 minutes toabout 1 hour, about 1 hour to about 48 hours, about 1 hour to about 24hours, about 1 hour to about 12 hours, about 1 hour to about 6 hours,about 1 hour to about 2 hours, about 2 hours to about 48 hours, about 2hours to about 24 hours, about 2 hours to about 12 hours, about 2 hoursto about 6 hours, about 6 hours to about 48 hours, about 6 hours toabout 24 hours, about 6 hours to about 12 hours, about 12 hours to about48 hours, about 12 hours to about 24 hours, or about 24 hours to about48 hours.

In some embodiments, the β-1,3/1,6-D-glucan is administered from about30 minutes to about 48 hours after a therapeutic procedure, for example,from about 30 minutes to about 48 hours, about 30 minutes to about 24hours, about 30 minutes to about 12 hours, about 30 minutes to about 6hours, about 30 minutes to about 2 hours, about 30 minutes to about 1hour, about 1 hour to about 48 hours, about 1 hour to about 24 hours,about 1 hour to about 12 hours, about 1 hour to about 6 hours, about 1hour to about 2 hours, about 2 hours to about 48 hours, about 2 hours toabout 24 hours, about 2 hours to about 12 hours, about 2 hours to about6 hours, about 6 hours to about 48 hours, about 6 hours to about 24hours, about 6 hours to about 12 hours, about 12 hours to about 48hours, about 12 hours to about 24 hours, or about 24 hours to about 48hours.

Pharmaceutical Formulations and Formulations

When employed as pharmaceuticals, the β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, provided herein can beadministered in the form of pharmaceutical compositions. In someembodiments, the β-1,3/1,6-D-glucan is administered in the form of apharmaceutical composition. In some embodiments, the polysaccharidecomprising the β-1,3/1,6-D-glucan is administered in the form of apharmaceutical composition. These compositions can be prepared asdescribed herein or elsewhere, and can be administered by a variety ofroutes, depending upon whether local or systemic treatment is desiredand upon the area to be treated. In some embodiments, including withoutlimitation for use in cosmetics, tonics, and health drinks, conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be included. In some embodiments, the compositions providedherein are suitable for oral administration. In some embodiments, thecompositions provided herein are suitable for parenteral administration.Parenteral administration includes intravenous, intraarterial,subcutaneous, intraperitoneal intramuscular or injection or infusion; orintracranial, (e.g., intrathecal or intraventricular, administration).Parenteral administration can be in the form of a single bolus dose, ormay be, for example, by a continuous perfusion pump.

Also provided are pharmaceutical compositions which contain, as theactive ingredient, the β-1,3/1,6-D-glucan provided herein in combinationwith one or more pharmaceutically acceptable carriers (e.g.,excipients). Also provided are pharmaceutical compositions whichcontain, as the active ingredient, the polysaccharide comprising theβ-1,3/1,6-D-glucan provided herein in combination with one or morepharmaceutically acceptable carriers (e.g., excipients). In making thecompositions provided herein, the active ingredient is typically mixedwith an excipient, diluted by an excipient or enclosed within such acarrier in the form of, for example, a capsule, sachet, paper, or othercontainer. When the excipient serves as a diluent, it can be a solid,semi-solid, or liquid material, which acts as a vehicle, carrier ormedium for the active ingredient. Thus, the compositions can be in theform of tablets, pills, powders, lozenges, sachets, cachets, elixirs,suspensions, creams, jelly, emulsions, solutions, syrups, soft and hardgelatin capsules, and sterile packaged powders.

Some examples of suitable excipients include, without limitation,lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia,calcium phosphate, alginates, tragacanth, gelatin, calcium silicate,microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water,syrup, and methyl cellulose. The formulations can additionally include,without limitation, lubricating agents such as talc, magnesium stearate,and mineral oil; wetting agents; emulsifying and suspending agents;preserving agents such as methyl- and propylhydroxy-benzoates;sweetening agents; flavoring agents, or combinations thereof.

The active compound can be effective over a wide dosage range and isgenerally administered in a therapeutically effective amount. It will beunderstood, however, that the amount of the compound actuallyadministered will usually be determined by a physician, according to therelevant circumstances, including the condition to be treated, thechosen route of administration, the actual compound administered, theage, weight, and response of the individual subject, the severity of thesubject's symptoms, and the like.

Accordingly, the present application provides methods of treating adisease of the digestive system described herein in a subject in needthereof, comprising administering to the subject a pharmaceuticalcomposition comprising β-1,3/1,6-D-glucan, or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptableexcipients.

The present application further provides methods of treating one or moresymptoms of a disease of the digestive system described herein in asubject in need thereof, comprising: a) identifying or having identifieda subject as exhibiting one or more symptoms of the disease of thedigestive system; and b) administering to the subject a pharmaceuticalcomposition comprising β-1,3/1,6-D-glucan, or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptableexcipients. In some embodiments, at least one of the symptoms isinflammation associated with the disease of the digestive system.

In some embodiments, the subject is identified or has been identified asexhibiting inflammation associated with a disease of the digestivesystem. In some embodiments, the subject is identified or has beenidentified as exhibiting inflammation associated with one or more organsof the digestive system. In some embodiments, the subject is identifiedor has been identified as exhibiting inflammation associated with one ormore organs associated with the digestive system. In some embodiments,the subject is identified or has been identified as exhibiting one ormore symptoms associated with a disease of the digestive system selectedfrom the group consisting of inflammatory bowel disease, Crohn'sdisease, and ulcerative colitis.

The present application further provides methods of treatingST-elevation myocardial infarction in a subject in need thereof,comprising administering to the subject a pharmaceutical compositioncomprising β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients.

The present application further provides methods of treating one or moresymptoms of ST-elevation myocardial infarction in a subject in needthereof, comprising: a) identifying or having identified a subject asexhibiting one or more symptoms of the ST-elevation myocardialinfarction; and b) administering to the subject a pharmaceuticalcomposition comprising β-1,3/1,6-D-glucan, or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptableexcipients. In some embodiments, at least one of the symptoms isinflammation associated with ST-elevation myocardial infarction.

In some embodiments, the pharmaceutical composition comprises about 10mg to about 1 g β-1,3/1,6-D-glucan, or a pharmaceutically acceptablesalt thereof, for example, about 10 mg to about 1 g, about 10 mg toabout 800 mg, about 10 mg to about 600 mg, about 10 mg to about 400 mg,about 10 mg to about 200 mg, about 10 mg to about 100 mg, about 10 mg toabout 50 mg, about 50 mg to about 1 g, about 50 mg to about 800 mg,about 50 mg to about 600 mg, about 50 mg to about 400 mg, about 50 mg toabout 200 mg, about 50 mg to about 100 mg, about 100 mg to about 1 g,about 100 mg to about 800 mg, about 100 mg to about 600 mg, about 100 mgto about 400 mg, about 100 mg to about 200 mg, about 200 mg to about 1g, about 200 mg to about 800 mg, about 200 mg to about 600 mg, about 200mg to about 400 mg, about 400 mg to about 1 g, about 400 mg to about 800mg, about 400 mg to about 600 mg, about 600 mg to about 1 g, about 600mg to about 800 mg, or about 800 mg to about 1 g. In some embodiments,the pharmaceutical composition comprises about 500 mg to about 600 mgβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof. Insome embodiments, the pharmaceutical composition comprises about 500 mgto about 550 mg β-1,3/1,6-D-glucan, or a pharmaceutically acceptablesalt thereof.

The present application further provides methods of treatinginflammatory bowel disease in a subject in need thereof, comprisingadministering to the subject a pharmaceutical composition comprisingabout 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.

The present application further provides methods of treating Crohn'sdisease in a subject in need thereof, comprising administering to thesubject a pharmaceutical composition comprising about 500 mg to about600 mg of β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients. In someembodiments, β-1,3/1,6-D-glucan is administered to the subject.

The present application further provides methods of treating ulcerativecolitis in a subject in need thereof, comprising administering to thesubject a pharmaceutical composition comprising about 500 mg to about600 mg of β-1,3/1,6-D-glucan, or a pharmaceutically acceptable saltthereof, and one or more pharmaceutically acceptable excipients.

In some embodiments, the pharmaceutical composition comprisesβ-1,3/1,6-D-glucan. In some embodiment, the pharmaceutical compositioncomprises the polysaccharide comprising the β-1,3/1,6-D-glucan providedherein.

Definitions

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active compound or pharmaceutical agent that elicits thebiological or medicinal response that is being sought in a tissue,system, animal, individual or human by a researcher, veterinarian,medical doctor or other clinician.

As used herein, the term “subject” or “patient,” used interchangeably,refers to any animal, including mammals, preferably mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,and most preferably humans.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) inhibiting the disease; for example, inhibiting a disease,condition or disorder in an individual who is experiencing or displayingthe pathology or symptomatology of the disease, condition or disorder(i.e., arresting further development of the pathology and/orsymptomatology); and (2) ameliorating the disease; for example,ameliorating a disease, condition or disorder in an individual who isexperiencing or displaying the pathology or symptomatology of thedisease, condition or disorder (i.e., reversing the pathology and/orsymptomatology) such as decreasing the severity of disease or reducingor alleviating one or more symptoms of the disease.

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” one or more cytokines with the β-1,3/1,6-D-glucandescribed herein includes the administration of the β-1,3/1,6-D-glucanto an individual or subject, such as a human, having one or more of thecytokines described herein, as well as, for example, introducingβ-1,3/1,6-D-glucan into a sample containing a cellular or purifiedpreparation containing one or more cytokines.

As used herein, “pharmaceutically acceptable salts” refers toderivatives of the disclosed compounds (e.g., β-1,3/1,6-D-glucan or thepolysaccharide comprising the β-1,3/1,6-D-glucan) wherein the parentcompound is modified by converting an existing acid or base moiety toits salt form. Examples of pharmaceutically acceptable salts include,but are not limited to, mineral or organic acid salts of basic residuessuch as amines; alkali or organic salts of acidic residues such ascarboxylic acids; and the like. The pharmaceutically acceptable salts ofthe present application include the conventional non-toxic salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. The pharmaceutically acceptable salts of the present applicationcan be synthesized from the parent compound which contains a basic oracidic moiety by conventional chemical methods. Generally, such saltscan be prepared by reacting the free acid or base forms of thesecompounds with a stoichiometric amount of the appropriate base or acidin water or in an organic solvent, or in a mixture of the two;generally, non-aqueous media such as diethyl ether, ethyl acetate,alcohols (e.g., methanol, ethanol, iso-propanol, or butanol), oracetonitrile are preferred. Lists of suitable salts can be found, forexample, in Remington's Pharmaceutical Sciences, 17th ed., MackPublishing Company, Easton, Pa., 1985, p. 1418 and Journal ofPharmaceutical Science, 66, 2 (1977), each of which is incorporatedherein by reference in its entirety.

EXAMPLES

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results. It is further appreciated that certain features of theinvention, which are, for clarity, described in the context of separateembodiments, can also be provided in combination in a single embodiment.Conversely, various features of the invention which are, for brevity,described in the context of a single embodiment, can also be providedseparately or in any suitable subcombination.

Example 1. Analysis of Polysaccharide Derived from Ganoderma lucidumMycelium

The polysaccharide derived from Ganoderma lucidum was analyzed accordingto the following methods:

Glycosyl Composition by GC-MS of TMS Derivatives of Methyl Glycosides

Glycosyl composition analysis was performed by combined gaschromatography/mass spectrometry (GC/MS) of the per-O-trimethylsilyl(TMS) derivatives of the monosaccharide methyl glycosides produced fromthe sample by acidic methanolysis as described previously by Santanderet al. Microbiology, 2013, 159:1471.

Glycosyl Linkage Analysis

For glycosyl linkage analysis, the samples were permethylated,depolymerized, reduced, and acetylated; and the resultant partiallymethylated alditol acetates (PMAAs) analyzed by gas chromatography-massspectrometry (GC-MS) as described by Heiss et al. Carbohydr. Res. 2009,344:915.

Size-Exclusion Chromatography (SEC)

The intact sample was loaded onto Superose12 GE-Amersham with optimalseparation range 1000-167,000 Da for dextrans, and the high MW fractionwas collected as “Peak 1” (i.e., Pk1) and was further analyzed forglycosyl composition and linkage analysis, and by 1D NMR-spectroscopy. Arepresentative SEC profile, as shown in FIG. 2A, shows that thepolysaccharide containing Peak 1 eluted at 8.9 min. Fractions werecollected between 7 and 12 min via multiple runs to isolate several mgof the polysaccharide. The obtained material was washed with nanopurewater and freeze dried for several times to remove any remaining bufferresidues. From SEC measurements, the molecular weight of Pk1 was foundto be approximately 3,800-4,000 kDa, as shown in FIG. 2B. The molecularweight reference standard used for the SEC molecular weight measurementwas 3,755 kDa. A second peak (Peak 2) was found to have a molecularweight of less than 5,000 Da, as shown in FIG. 2B and below in Table 1A.

TABLE 1A Peak Average Molecular Weight (Da) Peak area % Peak1 >3,755,000 54 Peak 2 <5,000 46

Tip-Sonication

To dissociate the polysaccharide Pk1 and to improve solubility, tipsonication was applied using Sonic Dismembrator 550 (Fisher Scientific)with micro tip.

NMR-Spectroscopy

The Peak 1 sample (˜5 mg) was deuterium-exchanged by lyophilization inD₂O. The total amount of the Peak 1 sample was dissolved in 0.6 mLDMSO-d₆ by incubating in 100° C. degree heat block for 1 h. D₂O (100 wasadded to the solution and the solution was transferred into a 5 mm ODNMR tube. 1D-Proton NMR of the sample in DMSO-d₆ and D₂O (6:1) wasacquired on a 600 MHz Varian Inova instrument at 65° C. Chemical shiftswere referenced to TMS (0 ppm).

Glycosyl composition analysis of the Peak 1 sample indicated thatGlucose (Glc) was the main residue at −97.4 mol %. Fuc, Gal, Xyl and Manwere detected in minor amounts as shown in FIG. 3 and below in Table 1B.Total carbohydrate weight percent was 75.8% as determined by compositionanalysis.

TABLE 1B Glycosyl Composition Analysis of the Peak 1 Sample Glycosylresidue Mass (μg) Mol %¹ Ribose (Rib) n.d. — Arabinose (Ara) n.d. —Rhamnose (Rha) n.d. — Fucose (Fuc) 1.8 0.6 Xylose (Xyl) 2.5 0.9Glucuronic Acid (GlcA) n.d. — Galacturonic acid (GalA) n.d. — Mannose(Man) 3.1 1.0 Galactose (Gal) 1.2 0.4 Glucose (Glc) 317.2  97.2 N-Acetyl Galactosamine (GalNAc) n.d. — N-Acetyl Glucosamine (GlcNAc)n.d. — N-Acetyl Mannosamine (ManNAc) n.d. — Σ= 325.9  100.1  ¹Values areexpressed as mole percent of total carbohydrate; n.d—not detected Thetotal mol percentage may not add to exactly 100% due to rounding.

Results of the glycosyl linkage analysis of the Peak 1 sample indicatedthe presence of variously linked glucosyl residues, as shown in FIG. 4and below in Table 2.

TABLE 2 Glycosyl Linkage Analysis of Peak 1 Sample Reten- tion Peak AreaResidue Time Area % Terminal Fucopyranosyl residue (t-Fuc) 11.2418435724 0.3 Terminal Xylopyranosyl residue (t-Xyl) 11.613 892156 0.0Terminal Mannopyranosyl residue (t-Man) 12.99 14055056 0.6 TerminalGlucopyranosyl residue (t-Glc) 13.15 551956030 22.9 2 linkedXylopyranosyl residue (2-Xyl) 15.166 6070200 0.3 3 linked Glucopyranosylresidue (3-Glc) 15.888 829730729 34.4 2 linked Mannopyranosyl residue(2-Man) 15.976 10988548 0.5 3 linked Mannopyranosyl residue (3-Man)15.976 10988548 0.5 2 linked Glucopyranosyl residue (2-Glc) 16.11515972737 0.7 4 linked Mannopyranosyl residue (4-Man) 16.472 9248469 0.46 linked Glucopyranosyl residue (6-Glc) 17.027 11464752 0.5 4 linkedGlucopyranosyl residue (4-Glc) 17.451 342058583 14.2 2,3 linkedMannopyranosyl residue (2,3- 18.146 2502773 0.1 Man) 3,4 linkedMannopyranosyl residue (3,4- 18.273 17648317 0.7 Man) 3,4 linkedGlucopyranosyl residue (3,4- 19.103 30574742 1.3 Glc) 2,3 linkedGlucopyranosyl residue (2,3- 19.307 50623330 2.1 Glc) 2,4 linkedGlucopyranosyl residue (2,4- 19.656 12432266 0.5 Glc) 3,6 linkedGlucopyranosyl residue (3,6- 20.281 379093860 15.7 Glc) 4,6 linkedGalactopyranosyl residue (4,6- 20.74 28991113 1.2 Gal) 2,3,4 linkedGlucopyranosyl residue 21.051 41995625 1.7 (2,3,4-Glc) 3,4,6 linkedGlucopyranosyl (3,4,6-Glc) 22.694 11627484 0.5 2,3,6 linkedGlucopyranosyl residue 24.554 24101514 1.0 (2,3,6-Glc) Total 2411452556100

Linkage results of the Peak 1 sample (Table 2) indicated the presence ofvariously linked glucosyl residues. The major linkages identified were:t-Glc (22.9% of total peak area), 3-Glc (34.4% of total peak area),4-Glc (>14.2% of total peak area) and 3,6-Glc (15.7% of total peak area)were detected. Furthermore, variously linked Man, Gal, Fuc, and Xylresidues were detected. The data corresponded with the results receivedfrom glycosyl composition analyses. The glycosyl composition resultswere calculated as mol % whereas linkage analysis results werecalculated as peak area. According to the results of linkage analysis,the total percentage of 3-Glc and 3,6-Glc together was approximately 76%of the total peak area. The linkage data in Table 2 also indicated thatthe ratio of 3-linked glucan (34.4%) to 3,6-Glc (15.7%) is 2:1.

The Peak 1 sample (i.e., Pk 1) isolated by preparative SEC was analyzedby 1D NMR spectroscopy, as shown in FIG. 5. 1D-Proton NMR-spectroscopicprofile of the Pk 1 in DMSO (DMSO-d₆ and D₂O 6:1 mixture) correlated tothe proton NMR profile of the β-Glucan USP (USP reference standard forβ-glucan) confirming that the main component of the Pk1 sample wasβ-1,3/1,6-glucan. Based on the combined linkage data and 1D-Proton NMRdata in dmso-d₆ the ratio between the 3-linked and 3,6-linkedbeta-glucan is 2:1 (Reference Standard: U.S. Pharmacopeia beta-glucanreference standard, Catalog Number 1048288, Lot FOK129).

TABLE 3 Integral values of the beta-glucan anomers of Pk 1 1D Proton NMRin DMSO-d₆ Peak, δ ppm Residue Normalized 4.57 3-β-Glc 2.06 4.25 6-β-Glc1.00

Based on these data, the structure of the beta-glucan was determined andis shown below:

Glucan Content

The total glucan content in the polysaccharide was measured using amushroom and yeast β-glucan assay kit (Megazyme). Table 4 showsrepresentative analysis data of the total glucan content in batches ofthe polysaccharide derived from mycelium of Ganoderma lucidum.

TABLE 4 Batch Number Total-glucan (%) α-glucan (%) β-glucan (%) 1 55.730.27 55.46 2 53.66 0.27 53.39 3 53.40 0.66 52.74 4 55.36 0.95 54.41 551.08 0.03 51.05 6 57.46 0.31 57.15 7 56.40 0.41 55.99 8 52.82 0.3252.50 9 53.49 0.84 52.65 10 50.85 0.54 50.31

Example 2. Treatment of Atherosclerosis Using β-1,3/1,6-D-glucan

This study was conducted to determine the effect of β-1,3/1,6-D-glucanin inhibiting the formation of atherosclerosis in patients with stableangina pectoris (SAP) and high risk patients (according to theFramingham score). To study the difference between the pre-test andpost-test treatment of 2 groups of patients (high-risk and (SAP)), adivergence test was performed using paired t test. An analysis was alsoperformed by comparing the equal treatment of the different yieldsbetween the test groups. The two groups of patients were classifiedaccording to the criteria described below:

Stable Angina Pectoris: Patients who experience typical anginacomplaints, such as discomfort in the chest area with substernalcrescendo characteristics that lasts approximately 10-15 minutes and isprovoked by emotional stress activity that disappear after rest ornitroglycerin administration, with ST-T wave change either ST segmentdepression of 0.5 mm or inverted T of 1 mm.

High Risk Patients/High Risk: The risk of coronary heart diseasecategorization for the next 10 years using a predictor of age, diabetesmellitus, smoking, blood pressure, total cholesterol level, HDL and LDLcholesterol using Framingham Score. Risk factor stratification forcoronary heart disease is declared high risk >20% according to ATP panelIII.

The present study has previously shown that β-1,3/1,6-D-glucansignificantly reduces the levels of MDA, hs-CRP, H2O2, totalcholesterol, and foam cells, and increase levels of HDL in experimentalanimals (Wistar strain Rattus novergicus) given a high-fat diet. Theβ-1,3/1,6-D-glucan also reduced the level of LDL, triglyceride, TNF-αand IL-6. The β-1,3/1,6-D-glucan, an anti-oxidant and anti-inflammatoryagent, is effective in reducing chronic inflammatory processes andoxidative stress that occur in the pathogenesis of cardiovasculardisease. The present study further demonstrated that no toxic effectswere found in both acute and subchronic studies on the administration ofβ-1,3/1,6-D-glucan to the experimental animals in any of the brain,kidney, liver, and lungs. The inhibition mechanism of the formation ofatherosclerosis was identified by measuring the following parameters inthe patient test groups: MDA, SOD, IL-6, hsCRP, TNF-α, TotalCholesterol, Triglycerides, Low Density Lipids (LDL), High DensityLipids (HDL), fasting glucose, HbA1c, systolic BP, diastolic BP and theratio of EPC-CEC.

Measurement of lipid profiles, IL-6, hsCRP, MDA, and SOD were performedusing human blood with pre-test and post-test treated blood collection.Measurement of lipid profile (HDL, LDL, triglycerides and totalcholesterol) was performed with a colorimetric spectrophotometry testusing Cobasmira tool. Measurement of IL-1, TNF Alpha, IL-6, hsCRP, MDA,SOD, CEC, NO, EPC, leptin and adiponectin was performed using an ELISAkit.

Data is presented in ±Standard Deviation (SD) mean form. To measure thedifference between the 2 groups of patients with High-Risk and SAPpre-test and post-test treatment, a divergence test was performed usingpaired t test. If the normality test indicated that the data was nothomogeneous, then a Mann Whitney test was used. To determine therelationship between the two treatment groups a statistical analysispaired t-test was used. If the normality test showed the datadistribution was not homogeneous, then a Wilcoxon rank statistical testwas used by applying SPSS version 17 (SPSS Inc). The difference ofP<0.05 was declared significant.

Patients were treated for 3 months with a dose of 1 capsule, 3×/day ascomplementary therapy. Each capsule contained 250 mg polysaccharide,which contained 180 mg β-1,3/1,6-D-glucan. The specific composition ofeach dose is shown below in Table 5.

TABLE 5 Component Amount per capsule Polysaccharides 250 mg (Consist ofglucose, galactose, arabinose, xylose, mannose and linked byβ-glycosidic linkages) β-D-Glucan content in 200 mg Polysaccharideβ-1,3/1,6-D-Glucan content in 180 mg β-D-Glucan Protein 10 mg As 0 ppmCd <1.5 ppm Pb <9 ppmSummary of Data for Patients having Stable Angina Pectoris (SAP)

FIGS. 6A-10 show data for pre- and post-administration of theβ-1,3/1,6-D-glucan for subjects in the SAP group. Table 6 shows asummary of divergence test in the SAP group assays.

TABLE 6 Variable Mean Sig (P) Pre IL-6 294.70 ± 123.28 0.000 Post IL-624.41 ± 21.45 Pre TNF alpha  11444 ± 2352.70 0.000 Post TNF alpha 476.13± 482.99 Pre CRP 20158.88 ± 8968.08  0.000 Post CRP   2092 ± 1437.16 PreMDA 95.63 ± 21.27 0.000 Post MDA 44.84 ± 50.95 Pre SOD 3.41 ± 0.46 0.001Post SOD 5.97 ± 4.19 Pre Adiponectin 33.33 ± 20.56 0.207 PostAdiponectin 21.65 ± 10.99 Pre Cholest 205.49 ± 48.49  0.081 Post Cholest182.11 ± 73.81  Pre HDL 46.20 ± 12.53 0.000 Post HDL 34.51 ± 16.66 PreLDL 126.17 ± 38.87  0.266 Post LDL 116.17 ± 54.16  Pre TG 122.37 ±62.04  0.974 Post TG 122.63 ± 96.54  Pre GLUC 22.88 ± 12.44 0.342 PostGLUC 7.47 ± 6.22 Pre HBA1C 7.68 ± 5.81 0.009 Post HBA1C 1.91 ± 2.52 PreCEC % Gated 7.87 ± 8.72 0.075 Post CEC % Gated 2.03 ± 3.05 Pre CEC %Total 2.52 ± 3.05 0.000 Post CEC % Total 0.71 ± 1.41 Pre EPC % Gated15.01 ± 8.39  0.000 Post EPC % Gated 5.43 ± 5.37 Pre EPC % Total 5.16 ±4.59 0.000 Post EPC % Total 1.19 ± 1.76 Pre NO % Gated 22.68 ± 12.440.000 Post NO % Gated 7.47 ± 6.22 Pre NO % Total 7.68 ± 5.81 0.000 PostNO % Total 1.91 ± 2.52 Pre Systolic 118.43 ± 47.07  0.225 Post Systolic108.71 ± 52.91  Pre Diastolic 72.86 ± 28.55 0.269 Post Diastolic 67.43 ±32.57

The following inflammatory markers showed a significant decrease:pretest IL-6 from 294.70±123.28 to 24.41±21.45 with p: 0.00; pretestTNF-alpha from 11444±2352.70 to 476.13±482.99 p: 0.00; and pretest CRPfrom 20158.88±8968.08±1437.16 to 2092 with p: 0.00. This was inaccordance with the results of the corresponding animal studies.

For MDA and SOD antioxidant markers, it was found that pretest of MDAlevel decreased from 95.63±21.27 to 44.84±50.95, and pretest of SODlevel increased from 3.41±0.46 to 5.97±4.19, which is in accordance withthe corresponding animal studies. The obtained pretest adiponectinmarker showed a decrease from 33.33±20.56 to 21.65±10.99. The result ofthis decline was affected by risk factors of the patient. HighAdiponectin level plays a role in the protective function ofhyperglycemia and dyslipidemia. The lipid profile showed that totalcholesterol level decreased significantly compared to other lipids.Pretest total cholesterol: from 205.49±48.49 to 182.11±73.81 p: 0:08.This is consistent with the results of the corresponding animal studiesdescribed above.

HbA1c level were lowered significantly even when the glucose level didnot show significant reduction. Pre-test HbA1c level decreased from7.68±5.81 to 1.91±2.52, with p=0.009, whereas pre-test glucose leveldecreased from 22.88±12.44 to 7.47±6.22, with p=0.342. CEC levelsdecreased from pretest results of 7.87±8.72 to 2.03±3.05, p=0.075, whilethe EPC, which is a biomarker for endothelial neovascularization andrepair, did not significantly increase, (from 15.01 to 5.43, withp=0.000). Without being bound by theory, this is likely due to riskfactors affecting the patients, including hypertension, smoking,dyslipidemia, and/or diabetes.

Blood pressure systolic and diastolic levels did not show a significantdecrease. Pretest systolic blood pressure decreased from 118.43±47.07 to108.71±52.91, with p=0.225 and pretest diastolic blood pressuredecreased from 72.86±28.55 to 67.43±32.57 with p=0.269.

Summary of Data for High Risk Patients

FIGS. 11A-15 show data for pre- and post-administration of theβ-1,3/1,6-D-glucan for subjects in the high risk group. Table 7 shows asummary of divergence test of the high risk group.

TABLE 7 Variable Mean Sig (P) Pre IL-6 279.75 ± 120.76 0.000 Post IL-629.32 ± 26.44 Pre TNF alpha 13447.84 ± 2199.46  0.000 Post TNF alpha544.85 ± 292.06 Pre CRP  25329 ± 8682.10 0.000 Post CRP 2659 ± 69  PreMDA 114.13 ± 24.56  0.000 Post MDA 36.84 ± 26.39 Pre SOD 3.12 ± 0.700.219 Post SOD 3.62 ± 4.26 Pre Adiponectin 24.74 ± 2.45  0.946 PostAdiponectin 35.09 ± 30.60 Pre Cholest 219.46 ± 49.49  0.193 Post Cholest201.43 ± 81.63  Pre HDL 52.03 ± 12.16 0.000 Post HDL 40.76 ± 19.31 PreLDL 132.24 ± 38.75  0.580 Post LDL 127.08 ± 54.19  Pre TG 128.57 ±62.13  0.201 Post TG 115.03 ± 63.80  Pre GLUC 89.78 ± 16.51 0.172 PostGLUC 91.81 ± 40.06 Pre HBA1C 5.86 ± 0.83 0.240 Post HBA1C 5.34 ± 1.66Pre CEC % Gated 6.96 ± 4.51 0.000 Post CEC % Gated 1.59 ± 1.52 Pre CEC %Total 2.07 ± 1.54 0.000 Post CEC % Total 0.6457 ± 0.66  Pre EPC % Gated13.06 ± 8.05  0.000 Post EPC % Gated 1.72 ± 2.24 Pre EPC % Total 3.28 ±3.66 0.010 Post EPC % Total 1.72 ± 2.24 Pre NO % Gated 20.03 ± 11.150.000 Post NO % Gated 6.61 ± 5.23 Pre NO % Total 5.35 ± 4.54 0.002 PostNO % Total 2.36 ± 2.62 Pre Systolic 130.14 ± 43.37  0.109 Post Systolic118.24 ± 55.68  Pre Diastolic   80 ± 25.74 0.102 Post Diastolic 73.24 ±33.85

The following inflammatory markers showed a significant decrease inpost-test results compared to pre-test results: pre-test IL-6 from279.75±120.76±26.44 to 29.32 with p: 0.000; pretest TNF-alpha from13447.84±2199.46 to 544.85±292.06, with p: 0.000; and pretest CRP from25329±8682.10 to 2659±89 with p: 0.000. This data corresponded to theresults from the animal data described above. For MDA and SODantioxidant markers it was found that pretest MDA level decreased from114.13±24.56 to 36.84±28.39 and pretest SOD increased from 0.70±3.12 to3.62±4.26. This data corresponded to the results from the animal datadescribed above.

The pre-test adiponectin marker increased from 24.74±2.45 to35.09±30.60. This increase in adiponectin corresponded with the theorythat high adiponectin levels play a role in protecting the vascularendothelium, particularly from dyslipidemia that damages vascularendothelium. The lipid profiles showed that total cholesterol leveldecreased significantly compared to other lipids; pretest totalcholesterol decreased from 219.46±49.49 to 201.43±81.63 with p: 0172.This data was consistent with the results obtained in the animal studiesdescribed above.

The HbA1c level decreased significantly even when the glucose level didnot decrease significantly. Pretest HbA1c levels decreased from5.86±0.83 to 5.34±1.88, with p=0.009, whereas pretest glucose level from89.78±18.51 increased to 91.81±40.06, with p=0.342. Pretest CEC leveldecreased from 6.96±4.51 to 1.59±1.52, p=0.000, while EPC did notincrease significantly: 13.06±8.05 to 1.72±2.24, with p=0.000. Withoutbeing bound by theory, this is likely due to risk factors affecting thepatients, including hypertension, smoking, dyslipidemia, and/ordiabetes.

Systolic and diastolic blood pressure levels did not show a significantdecrease. Pre-test systolic blood pressure decreased from 130.14±43.37to 118.24±55.68, with p=0.109 and pre-test diastolic blood pressuredecreased from 80±25.74 to 73.24±33.85 with p=0.102.

The data described above shows β-1,3/1,6-D-glucan anti-inflammatoryactivity, anti-oxidant activity, anti-lipid activity, andanti-endothelial dysfunction activity towards patients categorized inthe stable angina pectoris (SAP) and High Risk groups, and thatβ-1,3/1,6-D-glucan is able to act as secondary prevention for patientswith SAP and primary preventive for high risk subjects.

Example 3. Treatment of STEMI Using β-1,3/1,6-D-glucan

This study is an experimental clinical trial with human patients usingperspective randomized control with pre- and post-test design ofβ-1,3/1,6-D-glucan administration for 90 days with a hypothesis based onresults of the experiments described above in Example 2. The clinicaltrial is performed, for example, as shown in FIG. 16.

The targeted population is a representation of secondary prevention, apopulation with ST Elevation Myocardial Infarction with secondaryprevention, i.e. Stable Angina Pectoris (criteria according to ESC2014). STEMI is a clinical manifestation of atherosclerosischaracterized by total occlusion of the coronary arteries that lead tounmet needs of oxygen in tissues. The treatment population will bedivided into 2 groups, a control group and a treatment group. Bothgroups will receive different treatments (e.g., placebo for the controlgroup and β-1,3/1,6-D-glucan for the treatment group) and a measurablesignificant difference is expected. The results will verify the resultsof studies described above in Example 2. The population that willreceive β-1,3/1,6-D-glucan for 90 days will undergo patientstratification including physical examination, laboratory examinationCKMB, cardio troponin I (cTnI), and ECG. Patients participating in thestudy will be given informed consent of the study.

The marker results to be studied are anti-inflammatory (Hs-CRP, IL-6),anti-oxidant (MDA and SOD) and lipid profile (TC, TG, HDL, LDL),analysis of LV function, LVEF, stroke volumes, LV mass index, fractionalshortening, and wall segment analysis using echocardiograph that will bechecked before the beginning of β-1,3/1,6-D-glucan administration andafter the end of 90 days of administration of β-1,3/1,6-D-glucan. Theobtained data will be analyzed statistically using SPSS 17:00.

Measurement of lipid profile, IL-6, hsCRP, MDA, and SOD in human bloodis conducted through pre-test and post-test blood samples. Measurementof lipid profile (HDL, LDL, triglycerides and total cholesterol) isconducted through spectrophotometric colorimetric test using cobasmiratool. Measurement of IL-1, TNF-α, IL-6, hsCRP, MDA, and SOD is performedusing an ELISA kit.

Echocardiography is performed to assess LV function, LVEF, LV massindex, fractional shortening, and analytics wall segments.

Data is presented in the form of mean±SD. To compare the differencebetween the 2 groups of patients with STEMI who receives placebo andpatients with STEMI who received β-1,3/1,6-D-glucan treatment, differentpre-test and post-test tests are performed using paired t test. If thenormality test indicates that the data are not homogeneous, the MannWhitney will be applied. To determine the relationship between 2 groupsusing statistical paired t-test analysis, if the normality of datadistribution test is not homogeneous, the Wilcoxon rank statistical testwill be applied. Statistical calculations are done with SPSS version 17(SPSS Inc). Differences P of <0.05 is declared as significant.

Example 4. Treatment of Inflammatory Bowel Disease Usingβ-1,3/1,6-D-Glucan

Based on the pathogenesis of inflammatory bowel disease (IBD),β-1,3/1,6-D-glucan can be used as an immunomodulator as a complementarytherapy. For example, the β-1,3/1,6-D-glucan will modulate the activityof immune cells, so that the inflammatory process can be suppressed. Theβ-1,3/1,6-D-glucan is able to bind to the receptors of immune cells,including:

a. Dectin-1

b. Toll-like Receptor 2/4

C. Scavenger Receptor

d. Lactosylceramide

e. Complement Receptor-3 (CR-3)

f. Langerin Receptor

The results of the experiments described in Example 2 demonstrate thatβ-1,3/1,6-D-glucan has anti-inflammatory activity as evidenced by thedecrease in inflammatory biomarkers (e.g., TNF-α and IL-6) compared tobefore the patients are administered with β-1,3/1,6-D-glucan. In thepathogenesis of IBD, the production of TNF-α and IL-6 occurs as a resultof inflammatory processes; therefore, β-1,3/1,6-D-glucan would bebeneficial as an anti-inflammation agent in treating IBD, either aloneor in combination with other therapeutic agents. For example,β-1,3/1,6-D-glucan can be used as a complementary therapy for treatingUlcerative Colitis (UC) when combined with a standard therapy fortreating UC, such as 5-ASA (5-aminosalicylic acid) and glisodin (enzymesuperoxide dismutase/SOD). The addition of β-1,3/1,6-D-glucan instandard therapy provides improvements to the parameters observed. Uponadministration of β-1,3/1,6-D-glucan to subjects having IBD, thefollowing parameters will be measured: erythrocyte sedimentation rate,fecal calprotectin, fecal M2PK, and C-reactive protein. Additionalexamples of inflammatory parameters observed in UC are shown below inTable 8.

TABLE 8 Ulcerative Colitis Parameter Ulcerative Colitis ParameterObserved at One of International Observed by the Indonesian NO Studies(sumber: clinicaltrials.gov) Medical Doctors 1. C-Reactive Protein (CRP)C-Reactive Protein (CRP) 2. Interleukin-2 (IL-2) Erythrocytesedimentation rate 3. Interleukin-6 (IL-6) Stool Calprotectin 4. TumorNecrosis Factor - α Stool M2-Pyruvate Kinase (TNF-α) (M2-PK) 5. StoolCalprotectin 6. Inflammatory Bowel Diseases Questionnaire (IBDQ) 7.Subject Global Impression of Change (SGIC) 8. Physician's GlobalAssessment of Illness Severity (PGAS) 9. Ulcerative Colitis Symptoms:Pain 10. Ulcerative Colitis Symptoms: Stool Frequency 11. UlcerativeColitis Symptoms: Rectal Bleeding 12. Plasma Endocannabinoid Levels:2-arachidonylglycerol (2-AG) 13. Plasma Endocannabinoid Levels:anandamide (AEA) 14. Plasma Endocannabinoid Levels: oleoylethanolamide(OEA) 15. Clinical Body Weight Assessment

Example 5. Effect of β-1,3/1,6-D-glucan on Hematologic Profile

80 rats were randomly divided into 4 groups of female rats and 4 groupsof male rats (10 rats/group). The four groups of male and female ratswere subdivided into 3 treatment groups that received β-1,3/1,6-D-glucanat a dose of 300, 600, and 1200 mg/kg body weight (BW)/day for 90 days;and 1 control group. The following hematologic profile parameters weremeasured after 90 days:

-   -   Leukocytes    -   Platelets    -   Erythrocytes    -   Plateletcrit (PCT)    -   Mean corpuscular volume (MCV)    -   Mean corpuscular hemoglobin (MCH)    -   Red cell distribution width (RDW)

One-way test of the analysis of variance test (ANOVA) showed that therewas no significant difference in the value of leukocytes between thecontrol group and the treatment group receiving 3 variants of theβ-1,3/1,6-D-glucan dose. Significant differences occurred in the valueof erythrocytes and platelets, PCT, MCV, MCH, and RDW levels between thecontrol group and the three treatment groups, but the difference wasstill within the normal range. These data suggest thatβ-1,3/1,6-D-glucan with a dose of 300, 600, and 1200 mg/kg BW/day do nothave a harmful effect on blood components (hematological profile).

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

1. A method of treating a disease of the digestive system in a subjectin need thereof, comprising administering to the subject atherapeutically effective amount of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof.
 2. (canceled)
 3. The method ofclaim 1, wherein the disease of the digestive system is selected fromthe group consisting of inflammatory bowel disease, Crohn's disease, andulcerative colitis.
 4. (canceled)
 5. The method of claim 1, wherein themethod comprises administering about 500 mg to about 600 mgβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, tothe subject.
 6. (canceled)
 7. A method of treating a disease of thedigestive system in a subject in need thereof, comprising administeringto the subject a pharmaceutical composition comprisingβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, andone or more pharmaceutically acceptable excipients.
 8. (canceled)
 9. Themethod of claim 7, wherein the disease of the digestive system isselected from the group consisting of inflammatory bowel disease,Crohn's disease, and ulcerative colitis.
 10. (canceled)
 11. The methodof claim 7, wherein the pharmaceutical composition comprises about 500mg to about 600 mg of the β-1,3/1,6-D-glucan, or a pharmaceuticallyacceptable salt thereof.
 12. (canceled)
 13. The method of claim 1,wherein the disease of the digestive system is inflammatory boweldisease, and wherein the method comprises administering to the subjectabout 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof.
 14. The method of claim 1,wherein the disease of the digestive system is Crohn's disease, andwherein the method comprises administering to the subject about 500 mgto about 600 mg of β-1,3/1,6-D-glucan, or a pharmaceutically acceptablesalt thereof.
 15. The method of claim 1, wherein the disease of thedigestive system is ulcerative colitis, and wherein the method comprisesadministering to the subject about 500 mg to about 600 mg ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof. 16.(canceled)
 17. The method of claim 7, wherein the disease of thedigestive system is inflammatory bowel disease, and wherein the methodcomprises administering to the subject a pharmaceutical compositioncomprising about 500 mg to about 600 mg of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.
 18. The method of claim 7,wherein the disease of the digestive system is Crohn's disease, andwherein the method comprises administering to the subject apharmaceutical composition comprising about 500 mg to about 600 mg ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, andone or more pharmaceutically acceptable excipients.
 19. The method ofclaim 7, wherein the disease of the digestive system is ulcerativecolitis, wherein the method comprises administering to the subject apharmaceutical composition comprising about 500 mg to about 600 mg ofβ-1,3/1,6-D-glucan, or a pharmaceutically acceptable salt thereof, andone or more pharmaceutically acceptable excipients.
 20. (canceled)
 21. Amethod of treating one or more symptoms of a disease of the digestivesystem in a subject in need thereof, comprising: a) identifying orhaving identified a subject as exhibiting one or more symptoms of thedisease of the digestive system; and b) administering to the subject atherapeutically effective amount of β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof.
 22. (canceled)
 23. The methodof claim 21 or 22, wherein the subject is identified or has beenidentified as exhibiting one or more symptoms associated with a diseaseof the digestive system selected from the group consisting ofinflammatory bowel disease, Crohn's disease, and ulcerative colitis. 24.(canceled)
 25. The method of claim 21, wherein the method comprisesadministering about 500 mg to about 600 mg β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof, to the subject.
 26. (canceled)27. A method of treating one or more symptoms of a disease of thedigestive system in a subject in need thereof, comprising: a)identifying or having identified a subject as exhibiting one or moresymptoms of the disease of the digestive system; and b) administering tothe subject a pharmaceutical composition comprising β-1,3/1,6-D-glucan,or a pharmaceutically acceptable salt thereof, and one or morepharmaceutically acceptable excipients.
 28. (canceled)
 29. The method ofclaim 27, wherein the subject is identified or has been identified asexhibiting one or more symptoms associated with a disease of thedigestive system selected from the group consisting of inflammatorybowel disease, Crohn's disease, and ulcerative colitis.
 30. (canceled)31. The method of claim 27, wherein the pharmaceutical compositioncomprises about 500 mg to about 600 mg of the β-1,3/1,6-D-glucan, or apharmaceutically acceptable salt thereof.
 32. (canceled)
 33. The methodof claim 1, further comprising identifying or having identified thesubject as exhibiting abnormal expression of one or more cytokinesselected from interleukin-6, interleukin-10, tumor necrosis factor-α,and C-reactive protein, or any combination thereof.
 34. (canceled) 35.The method of claim 1, wherein the method further comprisesadministering one or more additional therapeutic agents. 36-37.(canceled)